U.S. patent number 6,719,923 [Application Number 10/168,876] was granted by the patent office on 2004-04-13 for paste, which can undergo screen printing for producing a porous polymer membrane for a biosensor.
This patent grant is currently assigned to Inverness Medical Limited. Invention is credited to Lucy Macgregor, Jerry McAleer, Alan McNeilage, Jamie Roders, Matthias Stiene, Birgit Von Tiedemann.
United States Patent |
6,719,923 |
Stiene , et al. |
April 13, 2004 |
Paste, which can undergo screen printing for producing a porous
polymer membrane for a biosensor
Abstract
The invention relates to a paste, which can undergo screen
printing, for producing a porous polymer membrane. Said paste
contains at least one polymer, one or more solvents for the polymer
having a boiling point of >100.degree. C., one or more
non-solvents for the polymers (pore-forming agents) having a higher
boiling point than that of the solvent(s), and contains a
hydrophilic viscosity modifier.
Inventors: |
Stiene; Matthias (Inverness,
GB), Von Tiedemann; Birgit (Inverness, GB),
Roders; Jamie (Inverness, GB), Macgregor; Lucy
(Inverness, GB), McAleer; Jerry (Grove,
GB), McNeilage; Alan (Inverness, GB) |
Assignee: |
Inverness Medical Limited
(GB)
|
Family
ID: |
7660463 |
Appl.
No.: |
10/168,876 |
Filed: |
November 4, 2002 |
PCT
Filed: |
October 18, 2001 |
PCT No.: |
PCT/EP01/12073 |
PCT
Pub. No.: |
WO02/32559 |
PCT
Pub. Date: |
April 25, 2002 |
Foreign Application Priority Data
|
|
|
|
|
Oct 19, 2000 [DE] |
|
|
100 52 066 |
|
Current U.S.
Class: |
252/511;
106/1.05; 106/162.7; 106/236; 106/237; 106/241; 106/287.1; 521/50;
524/261; 524/39; 524/442; 600/309; 600/319; 600/347; 600/365;
600/395 |
Current CPC
Class: |
B01D
67/0011 (20130101); B01D 67/0013 (20130101); B01D
69/122 (20130101); B01D 69/141 (20130101); B01D
71/16 (20130101); C12Q 1/002 (20130101); B01D
2323/06 (20130101); B01D 2323/12 (20130101) |
Current International
Class: |
B01D
69/00 (20060101); B01D 71/00 (20060101); B01D
71/16 (20060101); B01D 67/00 (20060101); B01D
69/12 (20060101); B01D 69/14 (20060101); C12Q
1/00 (20060101); G01N 27/40 (20060101); H01B
001/00 (); C08L 001/14 (); C23C 020/00 () |
Field of
Search: |
;252/511 ;524/39,261,442
;521/50 ;106/1.05,162.7,236,237,241,287.1
;600/309,319,347,365,395 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Acquah; Samuel A.
Claims
What is claimed is:
1. A screen-printable paste for producing a porous polymer
membrane, comprising at least one polymer, one or more solvents for
the polymer with a boiling point of >100.degree. C., one or more
nonsolvents (pore formers) for the polymer with a higher boiling
point than the solvent(s) and a hydrophilic viscosity modifier.
2. A screen-printable paste as claimed in claim 1, characterized in
that the difference of the boiling points of solvent and pore
former is at least 30.degree. C.
3. A screen-printable paste as claimed in claim 1, characterized in
that the paste comprises cellulose acetate as polymer.
4. A screen-printable paste as claimed in claim 3, characterized in
that the paste comprises 1,4-dioxane and/or
4-hydroxymethylpentanone and/or ethyl acetate as solvent.
5. A screen-printable paste as claimed in claim 4, characterized in
that the paste comprise a long-chain alcohol as pore former.
6. A screen-printable paste as claimed in claim 5, characterized in
that the paste comprises n-octanol and/or 2-methyl-2,4-pentanediol
as pore former.
7. A screen-printable paste as claimed in claim 6, characterized in
that n-octanol and/or 2-methyl-2,4-pentanediol is present in a
proportion of 5-20% by weight.
8. A screen-printable paste as claimed in claim 1, characterized in
that the paste comprises hydrophilic silica xerogel as viscosity
modifier.
9. A screen-printable paste as claimed in claim 8, characterized in
that the silica xerogel is present in a proportion of 1-10% by
weight.
10. A screen-printable paste as claimed in claim 1, characterized
in that the paste additionally comprises vinylpyrolidone/vinyl
acetate copolymer (PVP/VA) and/or polyvinylpyrolidone (PVP).
11. A screen-printable paste as claimed in claim 10, characterized
in that the PVP/VA or PVP is present in a proportion of 0.1% by
weight.
12. A screen-printable paste as claimed in claim 1, characterized
in that the paste additionally comprises one or more enzymes.
13. A method for producing a screen-printable paste, by producing a
mixture of one or more solvent(s) for a polymer and one or more
nonsolvent(s) for a polymer (pore former), mixing in the polymer
until a uniform suspension results, rolling the suspension until a
clear gel results, adding a hydrophilic viscosity modifier, and
rolling the mixture until the viscosity modifier is uniformly
distributed.
14. The use of the paste as claimed in claim 1, for producing a
porous polymer membrane.
15. The use as claimed in claim 14, where the polymer membrane is
introduced into a biosensor test strip.
16. The use as claimed in claim 15, characterized in that the
biosensor is designed for measuring the blood glucose
concentration.
17. The use as claimed in claim 15, characterized in that the
biosensor is designed for determining the value of the
hematocrit.
18. A porous polymer membrane produced from the screen-printable
paste as claimed in claim 14.
Description
The present invention relates to a screen-printable paste for
producing a porous polymer membrane which can be used in
electrochemical sensors, especially in electrochemical biosensors,
for integrated preparation of, in particular, whole blood
samples.
Biosensors are already in use in a large number of diagnostic
methods, for example in the determination of the concentration of
various factors in body fluids such as blood. The aim in this
connection is to have sensors which require no elaborate processing
of the (blood) sample but provide a rapid result simply by applying
the body fluid to a test strip. This entail a specific biochemical
reaction taking place, such as, for example, the enzymatic
conversion of the component to be determined, which then brings
about an electron transfer between different electrodes (working
and reference electrodes), and this can be determined
quantitatively.
The disadvantage of most or the known electrochemical biosensors is
that, on application of the blood to the region provided therefor
on the test strip, the biochemical reaction which takes place is
influenced by other constituents present in the blood, especially
the red blood corpuscles (erythrocytes). Thus, for example, when
the values of the hematocrit (=volume of the erythrocytes as a
proportion of the total amount of blood in vol.wt. %) are high, the
value measured for glucose using conventional blood glucose sensors
is lower than the actual value. This adverse affect arises from the
fact that the erythrocytes influence, through adsorption onto the
reactive layer of the biosensor, the diffusion of glucose into the
latter and to the electrode and reduce the measured signal.
To solve this problem, various membranes which are put on top of
the enzyme layer, which is disposed on the electrodes, of the test
strip in order to keep the erythrocytes away from this layer have
been proposed.
Thus, for example, U.S. Pat. No. 5,658,444 describes an erythrocyte
exclusion membrane for a sensor, which consists of a
water-insoluble, hydrophobic polymer, of a water-soluble
hydrophilic polymer and of an erythrocyte aggregating agent and is
produced by spraying onto the surface of the test strip.
One disadvantage of this membrane is that the membrane pore
diameter varies as a function of the spraying distance and spray
pressure. In addition, the spraying on of the membrane during
production of the rest strip means an additional operation which is
different from the production of the remainder of the test strip
and is therefore elaborate, which makes the production process
complicated and thus costly.
It is therefore an object of the present invention to provide a
paste for producing a porous membrane which does not have the
disadvantages mentioned since it can be applied during the
biosensor production process by a method which fits in with the
remaining procedure and is therefore cost-effective, and provides a
membrane of constant pore size.
This object is achieved by a paste for a porous polymer membrane as
claimed in claim 1. Advantageous developments are evident from
claims 2 to 18.
The invention is explained below by means of the figures, where
FIG. 1 shows diagrammatically the structure of a test strip with
the membrane of the invention,
FIG. 2 shows the theological characteristics of the paste of the
invention,
FIG. 3a shows an electron micrograph of a polymer membrane with
inadequately developed pore structure,
FIG. 3b shows an electron micrograph of the polymer membrane of the
invention with well developed pore structure,
FIG. 4 shows the results of measurement with two biosensors, one of
them being provided with a membrane of the invention, comparing as
the values of the hematocrit increase,
FIGS. 5a to 5d show the clinical performance on comparison of four
blood glucose sensors.
FIG. 1 depicts the structure of a test scrip with the polymer
membrane of the invention. An electrode arrangement 2 in the form
of a carbon layer, which in turn in partly covered by an insulation
3, is located on a polyester support material 1. An enzyme and
mediator layer 4 is, disposed on the region of the electrode layer
which is left free by the insulation. In the case of a blood
glucose sensor, this layer comprises, for example, the enzyme
glucose oxidase and the mediator Fe.sup.3+. The polymer membrane 5
of the invention as arranged above the enzyme and mediator layer 4.
The whole is covered by an adhesive layer 6 and a cover sheet
7.
In the mass production of biosensors, the screen printing method is
used for printing the various layers such as electrode, insulting
and enzyme layers. The present invention provides a membrane which
can be applied with the same technique. On the one hand, this has
the advantage that the same screen printing device can be used for
printing the membrane and thus throughout the sensor production
process, which involves enormous economic advantages in mass
production. On the other hand, it is possible to produce by the
screen printing method reproducibly a membrane of uniform thickness
and pore size, which is not ensured with the other methods such as
spincoating, dipping or spraying.
For it to be possible to apply the paste used to produce the
polymer membrane by screen printing, the solvent(s) present therein
for the polymer must have a boiling point which is as high as
possible (above 100.degree. C.) in order to avoid premature drying
of the material in the printing machine. In addition, the paste
comprises a nonsolvent for the polymer, which acts as pore former
and has a higher boiling point than the solvent(s) used.
The paste must moreover have a suitable viscosity (30 000-50 000
cpi) in order to ensure uniform flow through the screen during the
printing. The viscosity of the paste is preferably reduced on
exposure to shear forces, as depicted in the rheological
characteristics in FIG. 2.
The polymer preferably used in the paste of the invention is
cellulose acetate (50 kDa). It is preferably present in a
proportion of about 8% by weight in the screen-printable paste. In
addition, a further polymer which may be present is cellulose
nitrate in a proportion of up to 10% by weight.
Solvents which can be used for the polymer are, for example,
1,4-dioxane (boiling point 102.degree. C.) and/or
4-hydroxymethylpentanone (boiling point 165.degree. C.). A
preferred composition comprise 0-20% by weight, more preferably 20%
by weight, of 1,4-dioxane and 0-70% by weight, more preferably 56%
by weight, of 4-hydroxymethylpentanone, it being possible
alternatively to replace the 4-hydroxymethylpentanone by ethyl
acetate or ethylene glycol diacetate.
It has emerged that long-chain alcohols with a boiling point of
>150.degree. C. are suitable as pore formers for the
screen-printable membrane paste; preference is given to n-octanol,
which hap a boiling point of 196.degree. C., and/or
2-methyl-2,4-pentanediol (MPD), which has a boiling point of
197.degree. C.
The paste is somewhat more tolerant to evaporation of dioxane on
use of 2-metyl-2,4-pentanediol (MPD) as pore former. Moreover the
cellulose acetate remains in solution longer, which extends the
time during which the paste remains in a printable state. This
extended "pot life" makes it possible to produce larger batches of
constant quality.
The pore former should be present in a proportion of 5-20% by
weight, preferably 12-15% by weight.
The viscosity modifiers used are, for example, hydrophilic silica
xerogels or equivalent "fumed silicas", bentonite, clay, Natrosol
or carbon black. They should be added in a proportion of from 1 to
10% by weight to the screen-printable paste. Preference is given to
hydrophilic Cab-O-Sils (proprietary name for silica xerogels
marketed by the Cabot organization), such as Cab-O-Sil M5,
Cab-O-Sil H5, Cab-O-Sil LM50. Cab-O-Sil LM130, in a proportion of
4% by weight.
It is also possible to add further additives such as Tween 20,
Triton X, Silvet 7600 or 7280, lauryl sulfate (SDS), other
detergents, and polyols such as glycerol, or hydrophilic polymers
such as polyvinylpyrolidone (PVP) or vinylpyrolidone/vinyl acetate
copolymers (PVP/VA) to the paste of the invention.
Addition of one or more of these additives is not obligatory for
producing the membrane; however it has emerged that they may
improve the wetting of the membrane and speed up the sensor
response. Preference is given to the use of PVP/VA or PVP in a
proportion of 0.1% by weight in the screen-printable paste.
Moreover the addition of the additives Bioterge, polyethyleneimine,
BSA, dextran, dicyclohexyl phthalate, gelatin, sucrose and/or
biuret may improve the separation of erythrocytes and plasma.
It is additionally possible to add enzyme, for example glucose
oxidase, even to the cellulose acetate paste so that printing of
the enzyme layer can be dispensed with in the biosensor production
process.
After application of a uniform layer of the printing paste to a
suitable substrate, the membrane is formed during the drying
process. There is formation of a porous layer and not of a
continuous film, because the solvents used have a lower boiling
point than the pore former; the solvents evaporate correspondingly
quickly and the cellulose acetate polymer precipitates in the
remaining film of the pore former.
However, in connection with a biosensor, it is not permissible to
use just a high temperature in the drying process, because the
enzymes/proteins used are denatured it the temperatures are too
high. The best results were achieved in connection with a biosensor
for determining glucose in whole blood with a drying temperature of
about 70.degree. C. The boiling points of the solvents and pore
formers used should be selected correspondingly.
A crucial factor for the pore formation is the viscosity modifier
used, which forms a gel together with the pore former in order to
stabilize the polymer structure. With the substances used, the gel
in produced through the interaction between the OH groups of the
silica xerogel and the long-chain alcohol (e.g. octanol). The
amount and the distribution of the gel produced during the drying
process eventually determines the size and shape of the pores which
develop.
Without addition of a viscosity modifier there is formation of an
emulsion from the solvent and the pore former, because the pore
former is unable on its own to stabilize the polymer skeleton. The
result is a white, smooth and unstructured film with entrapped pore
former, which does not allow lateral liquid transport. By
comparison, a clear film is obtained when no pore former is used in
the paste.
If the amounts of viscosity modifier used are too small (<1% by
weight), the resulting membrane has an only inadequately developed
pore structure, as shown in FIG. 3a.
Since the various suitable viscosity modifiers have different
surface properties, the viscosity modifier can be selected
depending on the required membrane or the required biosensor. For
example, with high mechanical stress, e.g. with long printing times
or on printing of very thin layers with a high squeegee pressure,
the Cab-O-Sil H5 is "crushed". The surface then shows
microscopically sharp fracture edges which may lead to lysis of the
red blood cells.
This is an unwanted property for a blood glucose sensor because the
basic current of the sensor is increased thereby. On the other
hand, this effect can be optimized, and the plasma from cells be
utilized directly in the sensor for the electrochemical detection.
One practical example would be the examination of hemoglobin in
erythrocyte. In this case, the mediator of the biosensor, e.g.
potassium hexacyanoferrate (III), reacts with the Fe (II) group of
the hemoglobin, producing potassium hexacyanoferrate (II) which can
be determined directly at the electrode of the biosensor. An enzyme
like that in the case of glucose determination is unnecessary in
this came because the mediator reacts directly with the hemoglobin.
It is possible in this way in practice to determine the value of
the hematocrit for a patient with similar measuring equipment as in
the monitoring of blood glucose, making the time-consuming use of
capillary tubes and centrifuge unnecessary.
Cab-O-Sil LM 150 consists of smaller particles than H5, which are
therefore more stable and are not damaged by the mechanical stress
during the printing process. This viscosity modifier is therefore
most suitable for producing a membrane for blood glucose
sensors.
In accordance with the above statement, the difference in boiling
points between solvent and pore former is, besides the
stabilization of the polymer skeleton by the viscosity modifier,
important for the formation of a suitable membrane. The difference
should be about 30.degree. C. in this case, so that there is
formation in the drying process of a film which comprises a
sufficiently high concentration of pore former in which the
membrane polymer can precipitate. If the boiling point differences
are smaller the pore former starts to evaporate before a critical
ratio between solvent and pore former is reached, which brings
about the precipitation of the membrane polymer.
After the screen-printable paste with the composition described
previously has been printed, and the solvent has evaporated, there
is formation through deposition of the cellulose esters of a
membrane with an average pore size of from 0.1 to 2 .mu.M, it being
possible to influence the pore size by the amount of long-chain
alcohol used. An electron micrograph of the membrane is shown in
FIG. 3b. Since erythrocytes have an average size of 8 to 10 .mu.m,
the membrane keeps them away from the enzyme layer, while the
plasma can pass through unhindered. In addition, the membrane
contributes to the mechanical stability of the enzyme layer and
prevents the enzyme being detached from the electrode on
application of the blood sample and then no longer being available
for the electrochemical reaction.
FIG. 4 illustrates by means of a series of measurements the fact
that at a constant glucose concentration the test strip provided
with a membrane of the invention provides, in contrast to a test
strip without membrane, constant results as the values of the
hematocrit increase, whereas the response with the test strip
without membrane decreases as the erythrocyte concentration
increases. Because of the increased diffusion barrier between the
enzyme layer and the blood sample the response overall is somewhat
reduced in the case of the sensor with membrane.
The invention is illustrated by means of the following
examples.
Production of the Printing Paste:
In accordance with the ratios of amounts indicated in the following
examples, a mixture of the solvent (e.g. hydroxymethylpenanone,
dioxane) and the pore former (e.g. octanol, MPD) is produced to
ensure uniform distribution of the two substances. In the next
step, all the additives (e.g. PVP/VA) are added and dissolved, if
necessary with the aid of ultrasound. The membrane polymer
(cellulose actate 50 kDa) is then mixed rapidly with the previously
produced solvent until a uniform suspension results. This
suspension is rolled for 48 h in a closed container until a clear
gel results, and it is possible to add the viscosity modifier (e.g.
Cab-O-Sil) to this. The finished printing paste is rolled for a
further 24 h in order to ensure uniform distribution of the
viscosity modifier.
EXAMPLE 1
Polymer(s): Cellulose acetate (Mw 30 000) 7.5% by weight Solvent:
Ethylene glycol diacetate (b.p. 186.degree. C.) 65.5% by weight
Pore former: n-Decanol (b.p. 231.degree. C.) 25.0% by weight
Viscosity modifier: Cab-O-Sil M5 2.0% by weight
EXAMPLE 2
Polymer(s): Cellulose acetate (Mw 50 000) 8.0% by weight Solvents:
1,4-Dioxane (b.p. 102.degree. C.) 35.0% by weight Ethyl acetate
(b.p. 154.degree. C.) 35.0% by weight Pore former: n-Octanol (b.p.
196.degree. C.) 18.0% by weight Viscosity modifier: Cab-O-Sil M5
4.0% by weight
EXAMPLE 3
Polymer(s): Cellulose acetate (Mw 50 000) 8.0% by weight Solvents:
1,4-Dioxane (b.p. 102.degree. C.) 20.0% by weight
4-Hydroxymethylpentanone (b.p. 165.degree. C.) 56.0% by weight Pore
former: n-Octanol (b.p. 196.degree. C.) 12.0% by weight Viscosity
modifier: Cab-O-Sil M5 4.0% by weight Additives: PVP/VA 0.1% by
weight
EXAMPLE 4
Polymer(s): Cellulose acetate (Mw 50 000) 7.4% by weight Solvents:
1,4-Dioxane (b.p. 102.degree. C.) 18.5% by weight
4-Hydroxymethylpentanone (b.p. 165.degree. C.) 55.6% by weight Pore
former: 2-Methyl-2,4-pentanediol 14.8% by weight Viscosity
modifier: Cab-O-Sil M5 3.7% by weight Additives: PVP/VA 0.1% by
weight
FIG. 5 shows the clinical performance of blood glucose sensors
a) without polymer membrane
b) with polymer membrane (composition of Example 2)
c) with polymer membrane (composition of Example 3)
d) with polymer membrane (composition of Example 4).
In the comparative clinical investigations, the results of
measurement with the various types of sensors were compared with
the results of measurement by the reference method (YSI Model 2300
Stat Plus), and the percentage deviation was plotted against the
values of the hematocrit for the individual blood samples. The
result in the ideal case is a measurement line horizontal to the x
axis. The gradient of these measurement lines, which is shown in
Table 1, provides information about the interference of the
hematocrit with the sensor system used.
TABLE 1 Gradient of the measurement lines Gradient in % Type 1 (no
membrane) -0.8253 100% Type 2 (membrane from -0.4681 56% Example 2)
Type 3 (membrane from -0.2946 35% Example 3) Type 4 (membrane from
-0.0273 .sup. 3.3% Example 4)
The data unambiguously reveal the superior performance of the
sensor system with the preferred membrane (composition of Example
4). This improvement is achieved through the separation of whole
blood and plasma directly in front of the electrode, because the
Nernst diffusion layer in front of the electrode can no longer be
extended into the region with erythrocytes and therefore also can
no longer be influenced by different values of the hematocrit.
The following comparative examples describe printing pastes in
which there is no suitable accordance between the pore former, the
solvents and the viscosity modifier.
Comparative Example 1
Polymer(s): Cellulose acetate (Mw 50 000) 8.0% by weight Solvent:
Ethylene glycol diacetate (b.p. 186.degree. C.) 76.0% by weight
Pore former: n-Octanol (b.p. 196.degree. C.) 12.0% by weight
Viscosity modifier: Cab-O-Sil M5 (hydrophilic) 4.0% by weight
Additives: PVP/VA 0.1% by weight
Comparative Example 2
Polymer(s): Cellulose acetate (Mw 50 000) 8.0% by weight Solvents:
1,4-Dioxane (b.p. 102.degree. C.) 20.0% by weight
4-Hydroxymethylpentanone (b.p. 165.degree. C.) 56.0% by weight Pore
former: n-Octanol (b.p 196.degree. C.) 12.0% by weight Viscosity
modifier: Cab-O-Sil TS720 (hydrophobic) 4.0% by weight Additives:
PVP/VA 0.1% by weight
Comparative Example 3
Polymer(s): Cellulose acetate propionate 8.0% by weight (Mw 75 000)
Solvents: 1,4-Dioxane (b.p. 102.degree. C.) 20.0% by weight
4-Hydroxymethylpentanone (b.p. 165.degree. C.) 56.0% by weight Pore
former: n-Octanol (b.p. 196.degree. C.) 12.0% by weight Viscosity
modifier: Cab-O-Sil M5 (hydrophilic) 4.0% by weight Additives:
PVP/VA 0.1% by weight
In Comparative Example 1 there is no formation of a porous membrane
because the difference between the boiling points of the solvent
(ethylene glycol diacetate) and pore former (n-octanol) used in the
printing paste is too small. If, by contrast, n-decanol is used as
pore former (as described in Example 1), a porous membrane is
obtained after the drying process because the boiling point between
the solvent and the pore former is sufficiently large.
In Comparative Example 2 there is only inadequate gel formation
between the pore former and the viscosity modifier, because of the
use of hydrophobic Cab-O-Sil which is unable to react with the OH
groups of the pore former, and thus there is inadequate
stabilization of the polymer skeleton. This impedes the formation
of a porous membrane.
No porous membrane is formed in Comparative Example 3 either, where
the solubility of the polymer used (cellulose acetate propionate)
in the pore is too high.
* * * * *